Cardiovascular Research

Cardiovascular Research 1999 41(3):654-662; doi:10.1016/S0008-6363(98)00275-2
© 1999 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Cheng, T.H. Articles by Chen, J.J. Search for Related Content PubMed PubMed Citation Articles by Cheng, T.H. Articles by Chen, J.J. Social Bookmarking          What's this?

Copyright © 1999, European Society of Cardiology

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes

T.H. Cheng^a, N.L. Shih^b, S.Y. Chen^b,1,1, D.L. Wang^b and J.J. Chen^b,c,*

^aGraduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC
^bCardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529 Taiwan, ROC
^cDepartment of Internal Medicine, Medical College of National Taiwan University, Taipei, Taiwan, ROC

^* Corresponding author. Tel.: +886-2-2789-9135; Fax: +886-2-2782-9143.

Received 19 December 1997; accepted 27 July 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. Methods: Intracellular ROS levels were measured with fluorescence probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. Results: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H₂O₂ treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (–2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H₂O₂ were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21^ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. Conclusions: These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses in cardiomyocytes.

KEYWORDS Endothelin-1; Reactive oxygen species; c-fos; Gene expression; Ras; Rat, Cardiomyocyte

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Endothelin-1 (Et-1) is a potent 21-amino acid long vasopressor secreted from endothelial cells [1]. Apart from its constrictive activity on vascular smooth muscle, Et-1 has been demonstrated to stimulate hypertrophic growth in cardiomyocytes [2, 3]. Et-1 can induce a transient increase in expression of immediate early genes including c-fos and early growth response-1 (egr-1) [4]. It has been reported that Et-1 activates a phosphorylation cascade of protein kinases including Raf-1 and the extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway in cardiac myocytes [3]. Et-1 also activates phosphorylation cascade of MAPK/Jun amino-terminal kinase (JNK) in cardiac myocytes [5, 6]. Both ERK and JNK can phosphorylate Elk-1, one of several ternary complex factors, and resulting in an increase of c-fos transcription [7–9]. c-fos and c-jun make the heterodimer complex of activating protein-1 (AP-1), which preferentially binds to many genes that have 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE) in their promoter region [10].

Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers to modulate signaling pathways [11]. ROS encompass many oxygen species including singlet oxygen, superoxide, hydrogen peroxide (H₂O₂) and hydroxyl radicals that are produced by virtually every type of cell [12]. ROS, when present in excess, are toxic to cells due to the potential damage they can cause to cellular components. However, there is growing evidence that ROS at low concentration serve as signaling molecules, providing a chemical link between alterations in intracellular redox status and responsive changes in the structure and function of transcription factors [12]. The changes of intracellular ROS have been detected in a variety of cells stimulated with cytokines, growth factors, agonists of receptor [12]. Studies have also demonstrated that Et-1 treatment can induce ROS levels in various cells [13, 14]. However, the origin of ROS and their signaling roles contributing to cellular responses in cardiomyocytes have not been well characterized.

The small G protein p21^ras (Ras) is a potent regulator of myogenic cell growth and differentiation. Ras has been demonstrated to play a role in Et-1-mediated c-fos activity in cardiomyocytes [15]. Ras has been implicated to play a major role in the genesis of cardiac hypertrophy by activating the Raf-1/ERK pathway [16, 17]. It has been reported that Ras contributes to the induction of the c-fos by Et-1 in mesangial cells [18]. Recent findings further indicate that Ras is involved in the ROS generation in fibroblasts [19, 20]. Furthermore, Et-1 has also been reported to increase the ROS levels in broncho-alveolar cells [13]. The c-fos gene has been shown to be induced by Et-1 [21]or oxidants [22]in cardiomyocytes. However, the role of Ras in the generation of ROS and their relation to c-fos expression in the Et-1-treated cardiomyocytes have not been determined. Since ROS can act as second messengers, the roles of ROS in cardiomyocytes upon Et-1 treatment have thus been studied. In the present study, we clearly demonstrated that ROS mediate c-fos gene induction via Ras pathway in Et-1 treated cardiomyocytes.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Reagents
Endothelin-1, BQ-485 and BQ-788 were purchased from Calbiochem (San Diego, CA, USA). N-Acetylcystein (NAC), catalase and other chemicals were purchased from Sigma (St. Louis, MO, USA).

2.2 Cardiomyocytes culture
Primary cultures of neonatal rat ventricular myocytes were prepared as previously described [2]. The rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Briefly, ventricles from 1- to 2-day-old neonatal Sprague–Dawley rats were cut into chunks of approximately 1 mm³ using scissors and subjected to trypsin (0.125%, Gibco) digestion in phosphate-buffered saline (PBS). Trypsin digested cells were collected by centrifugation at 1200 rpm for 5 min. The cell pellet was resuspended in a medium containing 80% F10 nutrient mixture, 20% fetal calf serum, penicillin (100 U/ml) and streptomycin (68.6 µmol/l) and plated into a petri dish for 2.5 h. The suspended nonattached myocytes in the medium were collected and plated on a 100-mm diameter culture dishes with cell density at 1·10⁷ cells/dish. After 2 days in culture, cells were transferred to medium containing 90% DMEM nutrient mixture, 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (68.6 µmol/l). Myocytes cultures thus obtained were >80% pure as revealed by their contractile characteristics under light microscopy. Serum-containing medium from these cultured myocytes was replaced with serum-free medium and exposed to agents as indicated. For measurement of intracellular ROS generation, cardiomyocytes were plated to 30-mm diameter culture dishes with low cell density (1·10⁴ cells/dish) to monitor intracellular oxidation in single cardiomyocyte.

2.3 Transfections
For the transient transfections, cardiac cells were transfected with different expression vectors by the calcium phosphate method [23]. The DNA–CaPO₄ precipitates in each dish (10 ml) contained the following if not specifically identified: 15 µg c-fosCAT reporter plasmid; 1 or 5 µg RasL61; 5 µg RasN17; 5 µg pSV-β-galactosidase plasmid; and variable amounts of pSR plasmid vector to adjust for total DNA. Briefly, cardiomyocytes were maintained in culture for 48 h prior to transfection. The indicated expression vectors were mixed with calcium phosphate and immediately added to the cardiomyocyte cell culture. After incubation for 5 h, cells were then washed three times with PBS and incubated with 10% serum DMEM. After 24 h, cells were washed with serum-free medium and incubated in the same medium for an additional 24 h. Cells were then treated with different agents. To correct for transfection efficiency, 5 µg of pSV-β-galactosidase plasmid, which contains a β-galactosidase gene driven by the simian virus 40 promoter and enhancer, was cotransfected into cells. The c-fos CAT vector included 5' flanking regions of a 2.25 kb EcoR I-Nae I fragment containing upstream sequences and the promoter of the c-fos gene [24]. The constitutively activated Ras (RasL61) and the dominant-negative Ras (RasN17) plasmids have been previously reported [25]and were kindly provided by J.J. Shyy, (University of California, San Diego, CA, USA). In some experiments, RasL61 or RasN17 were transfected in the absence or presence of c-fosCAT. The empty vector pSR was transfected and used as a control. Except for routine β-galactosidase activity, cells were constantly collected for measuring intracellular ROS, RNA levels and CAT activities as described below.

2.4 Assay of intracellular ROS
Intracellular ROS production was measured by using a fluorescent dye, 2',7'-dichlorofluorescin diacetate (DCF-DA) (Molecular Probes, Eugene, OR, USA) with the ACAS Interactive Laser Cytometer (Meridian Instruments, Okemos, MI, USA). DCF-DA is a membrane-permeable compound. After intracellular deacetylation, DCF-DA forms a nonfluorescent product (DCF), which upon oxidation is transformed into fluorescent DCF [26]. A 10 mmol/l stock solution of DCF-DA was prepared in ethanol on a daily basis and diluted to a final concentration of 10 µmol/l just before the experiments. Myocytes with or without transfection were preincubated with 10 µmol/l DCF-DA in DMEM for 30 min at 37°C prior to treatment. Cells after dye exposure were rinsed with normal Tyrode solution. The cells were maintained in normal Tyrode solution and examined with the Laser Cytometer at 37°C. Excitation of DCF was achieved using the 488-nm line of a 20-mW argon-ion laser. The emission above 515-nm is quantitated from two dimensional image scans generated by a 1 micron laser beam and an X–Y scanning stage. A fluorescence value from single cells can be obtained. To provide a valid comparison, the same acquisition parameters were used for all observations. Quantification of the levels of DCF fluorescence was assessed on a relative scale from 0–4000 units. Baseline values from unstimulated or pSR-tranfected cells were used as control values to compare with Et-1-stimulated or Ras-transfected cells. Values represent means±S.E.M. of DCF fluorescence from at least 20 randomly selected cells. This experiment was repeated for at least six times to avoid the transfection variation.

2.5 RNA isolation and northern hybridization
Total RNA was obtained by using guanidine thiocyanate as described previously [2]. Total RNA was collected and examined by mini-gel agarose electrophoresis. Ten micrograms of RNA was mixed with loading buffer containing ethidium bromide. The sample mixture was loaded and separated on 1% agarose gels containing 3.7% formaldehyde. RNA was transferred onto Nytran membrane (Schleicher & Schuell, Germany) by a vacuum blotting system (VacuGene XL, Pharmacia, Sweden) and immobilized by ultraviolet irradiation. A 2137 bp EcoR I/EcoR I fragment containing rat c-fos DNA [27]was used as the c-fos probe and was labeled by random priming with [³²P]-dCTP using the random primer DNA labeling kit (Amersham, Buckinghamshire, UK). Individual blot was subsequently washed and reprobed with 18S ribosomal RNA cDNA probe (obtained from American Type Culture Collection) to normalize differences in loading and/or transfer to the filter. Autoradiographic results were scanned with a Bio-Rad densitometer.

2.6 Chloramphenicol acetyltransferase assays and β-galactosidase assays
The chloramphenicol acetyltransferase (CAT) and β-galactosidase assays were performed as previously described [2]. The relative CAT activity was corrected by normalizing the respective CAT value to that of β-galactosidase activity. Cotransfected β-galactosidase activity varied by <10% within a given experiment and was not affected by any of the experimental manipulations described. As positive and negative controls, pBLCAT2 (with thymidine kinase promoter) and pBLCAT3 (without promoter) were included in every assay [23].

2.7 Statistical analysis
Data were presented as mean±S.E.M. Statistical analysis was performed using one-way analysis of variance (ANOVA) or an unpaired Student’s t-test. The significance level was set at 0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Et-1 induced intracellular ROS levels in cardiomyocytes
Intracellular generation of ROS in cardiomyocytes was analyzed by the fluorescent product (DCF), a peroxidative product of DCF-DA with laser-scanning confocal microscopy. Exposure of cardiomyocytes to Et-1 (10 nmol/l) rapidly induced DCF fluorescence with a maximal increase of 2-fold at 15 min after stimulation (Fig. 1). This ROS induction was shown to be an Et-1 concentration dependent since ROS level appeared to reach plateau after treating with 10 nmol/l of Et-1 (Fig. 2A). To specify which Et-1 receptor subtype that was responsible for the generation of ROS in cardiomyocytes, we pretreated cardiomyocytes with Et-1 receptor type A antagonist (BQ485) or receptor type B antagonist (BQ788) before the Et-1 exposure. As shown in Fig. 2B, BQ485 (100 nmol/l) significantly inhibited the Et-1-induced generation of ROS. In contrast, BQ788 (100 nmol/l) had no significant effect on Et-1-induced generation of ROS. These results suggest that an increase of intracellular ROS levels upon Et-1 treatment is mediated mainly through the binding of Et-1 to Et-1 receptor type A.

View larger version (67K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Et-1 induced ROS generation in cardiomyocytes. (A) Cardiomyocytes treated with Et-1 increased intracellular ROS levels as revealed by fluorescent intensities of DCF. Cardiomyocytes preincubated with DCF-DA for 30 min and then treated with Et-1 for the indicated times (min). Fluorescence intensity was analyzed with confocal laser cytometer. (B) Cardiomyocytes were photographied by differential interference contrast microscopy. (C) Time course of Et-1-induced ROS generation in cardiomyocytes as revealed by DCF fluorescence. About 20 randomly selected cells were used for determination of each point. The results show mean±S.E.M. *P<0.05 vs. control untreated cells. The experiment was repeated six times with reproducible results.

 

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Et-1-induced ROS levels were attenuated by pretreatment of cardiomyocytes with Et-1 antagonists or antioxidant. (A) Et-1 induced ROS generation in a dose-dependent manner. (B) Cardiomyocytes from either control (C; column 1) or treated with Et-1 (10 nmol/l) in the absence (column 2) or presence of 100 nmol/l BQ485 (column 3), 100 nmol/l BQ788 (column 4), 10 mmol/l NAC (column 5) or 350 U/ml catalase (column 6) for 15 min. H2O2 (25 µmol/l) treated cells (column 7) are shown as positive controls. Fluorescence intensities of cells are shown as relative intensity of experimental groups compared to untreated control cells. At least 20 randomly selected cells were used for determination of each experiment. The results show mean±S.E.M. *P<0.05 vs. control; #P<0.05 vs. Et-1 treated cells. The experiment was repeated six times with reproducible results.

 
To examine whether the ROS were responsible for the oxidation of DCF in cardiomycytes, we examined whether catalase, an enzyme that specifically hydrolyses H₂O₂, was able to prevent the oxidation of DCF. Cardiomyocytes were treated with Et-1 in the presence of catalase (350 U/ml). The addition of catalase to cultured cardiomyocytes completely inhibited Et-1-induced ROS levels as measured at 15 min after Et-1 treatment (Fig. 2B). NAC, a glutathione precursor and a free radical scavenger, also inhibited the increase in Et-1-induced intracellular ROS levels (Fig. 2B). These findings clearly demonstrate that Et-1 treatment to cardiomyocytes increases intracellular ROS levels.

3.2 Inhibitory effect of antioxidants on the expression of c-fos gene by Et-1
We then investigated whether ROS generated by Et-1 play a role in gene expression. Previous studies demonstrated that ROS stimulated the expression of immediate early response genes such as c-fos, c-jun, c-myc and egr-1 [28]. Of these genes, c-fos was found to respond to both Et-1 [4, 21]and H₂O₂ [22]treatment in cardiomyocytes. The c-fos mRNA levels were shown by Northern blotting at various time intervals after treating cardiomyocytes with either Et-1 (10 nmol/l) or H₂O₂ (25 µmol/l) (Fig. 3). As shown, the serum-deprived cultured cardiomyocytes incubated with Et-1 (10 nmol/l) rapidly increased c-fos mRNA levels within 10 min of Et-1 exposure. This induction reached a maximal level at 30 min, i.e., an 11-fold induction vs. control cells (Fig. 3A). Consistently, when cardiomyocytes were exposed to H₂O₂ (Fig. 3A), a time-dependent increase of c-fos mRNA levels was noticed. The H₂O₂ treatment for 60 min induced c-fos mRNA levels in cardiomyocytes about 13-fold as compared with controls (Fig. 3A). All these results suggest that Et-1-induced c-fos mRNA in cardiomyocytes are modulated through ROS generated during Et-1 exposure.

View larger version (57K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Antioxidant inhibited Et-1- or H2O2- induced c-fos mRNA in cardiomyocytes. (A) Time course study of c-fos mRNA induction by Et-1 and H2O2. Cardiomyocytes were from untreated controls (lane1 and 5) or from Et-1 (10 nmol/l, lane 2–4) or H2O2 (25 µmol/l, lane 6–8) treatment for various time intervals. (B) Inhibition of Et-1-induced c-fos mRNA by antioxidant. Cardiomyocytes were either untreated controls (lane 1), or treated with Et-1 (10 nmol/l) in the absence (lane 2) or presence of 10 mmol/l NAC (lane 3) or 350 U/ml catalase (lane 4) for 30 min. For H2O2 studies, cells were treated with H2O2 (25 µmol/l) in the absence (lane 5) or presence of NAC (lane 6) or catalase (lane 7) for 60 min. Cells treated with PMA (0.8 µmol/l; lane 8) for 30 min were used as positive controls. Total RNA was extracted and Northern hybridization was performed with 32P-labeled rat c-fos as probe. 18S ribosomal RNA was used to normalize the RNA applied in each lane. Data was presented as folds increase of experimantal groups compared to untreated controls. *P<0.05 vs. control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. H2O2 alone. Results are shown as mean±S.E.M. from five experiments in time course study and three experiments in panel B with reproducible results.

 
We then examined the effects of antioxidant or free radical scavenger, i.e. catalase and NAC, on the induction of c-fos by Et-1. As shown in Fig. 3B both Et-1 (10 nmol/l) and H₂O₂ (25 µmol/l) increased c-fos mRNA levels in cardiomyocytes. However, the addition of catalase (350 U/ml) and NAC (10 mmol/l) 30 min prior to the Et-1 and H₂O₂ treatment abolished the c-fos gene induction (Fig. 3B). Cardiomyocytes treated with PMA (phorbol 12-myristate 13-acetate; 0.8 µmol/l) for 30 min as positive controls induced c-fos mRNA levels about 21-folds as compared with controls (Fig. 3B). However, the c-fos induction by PMA was only partially inhibited after these antioxidants treatment (data not shown) indicating that the inhibitory effect of antioxidants was specific for Et-1-induced c-fos gene expression. These findings are consistent with the earlier results of ROS induction in cardiomyocytes after Et-1 treatment (Fig. 2). Taken together, these results suggest that Et-1-induced ROS levels are involved in the c-fos gene expression since antioxidant pretreatment of cardiomyocytes inhibits this c-fos induction.

3.3 ROS modulated Et-1-increased c-fos transcriptional activity
To further determine whether the ROS modulating the Et-1-induced c-fos expression was a transcriptional event, a c-fos promoter activity was examined by using a reporter gene construct containing c-fos promoter region (–2.25 kb) and reporter gene chloramphenicol acetyltransferase (CAT). This chimera was transfected into cardiomyocytes followed by Et-1 or H₂O₂ stimulation. As demonstrated in Fig. 4A, either catalase or NAC alone had no effect on the basal c-fos promoter activity. However, cardiomyocytes treated with Et-1 (10 nmol/l) and H₂O₂ (25 µmol/l) for 24 h led to a 2.7- and 2.1-fold increase in CAT activity, respectively, as compared to control unstimulated cells (Fig. 4B). In the presence of catalase (or NAC), Et-1- or H₂O₂-induced c-fos promoter activities were significantly inhibited. These data clearly indicate that ROS modulate the increase of c-fos promoter activity in cardiomyocytes after Et-1 or H₂O₂ treatment.

View larger version (55K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Antioxidant inhibited Et-1- or H2O2- induced c-fos promoter activity. (A) Cardiomyocytes were transfected with 15 µg of c-fosCAT reporter gene construct for 24 h. Some cells were pretreated with NAC or catalase for 30 min. Cardiomycytes were then treated with Et-1 (10 nmol/l) or H2O2 (25 µmol/l) for 24 h. PMA-treated cells are shown as positive controls. CAT2 (lane 11) and CAT3 (lane 12) are shown as positive and negative controls of a CAT assay system. Cells were harvested and CAT assays were performed as described in Section 2. (B) CAT activities are shown as percentage incorporation after normalizing to that of β-galactosidase activities. The results are shown as mean±S.E.M. *P<0.05 vs. control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. H2O2 alone. The experiment was repeated three times with reproducible results.

 
3.4 Ras mediated Et-1-induced ROS generation and c-fos expression
Ras has been demonstrated to be involved in the intracellular ROS generation in fibroblasts [20]. In order to study the role of Ras in the Et-1-induced ROS generation, expression plasmid encoding a constitutively activated Ras protein (RasL61) was used to transfect into cardiomyocytes. RasL61 is an active form of p21^ras in which the Gln-61 in the wild type has been replaced by Leu [25]. As shown in Fig. 5A, RasL61 (5 µg) transfection into cardiomyocytes led to a 4.5-fold increase in intracellular ROS levels as compared to control pSR-transfected cells. pSR-transfected cardiomyocytes after Et-1 treatment increased intracellular ROS levels. In contrast, cardiomyocytes transfected with dominant negative Ras mutant (RasN17; 5 µg), a dominant negative mutant of Ras in which Ser-17 in the wild type has been replaced by Asn so that the affinity to GTP is dramatically reduced [25], abolished the Et-1-induced ROS production (Fig. 5A). To understand whether these Ras-mediated ROS were involved in the Et-1-induced c-fos expression, Ras-transfected cells were analyzed for their c-fos mRNA levels. Transient expression of RasL61 (5 µg) for 48 h significantly increased the c-fos mRNA (Fig. 5B). The treatment of catalase 350 U/ml for 24 h only partially inhibited the RasL61-stimulated c-fos gene expression. Compared to control pSR-transfected cells, c-fos induction by Et-1 was abolished in cells transfected with RasN17 (5 µg), but not in those that received H₂O₂ (25 µmol/l) treatment for 30 min (Fig. 5B). To further confirm that Ras mediated Et-1-induced c-fos promoter activity, we cotransfected c- fosCAT reporter gene with RasL61 (or RasN17). Cotransfections of the c-fosCAT reporter gene with RasL61 (5 µg) stimulated c-fosCAT activity by 3.8-fold as compared with control pSR-transfected cells (Fig. 6). The treatment of catalase 350 U/ml for 24 h partially inhibited the RasL61 stimulated c-fos promoter activity. Cotransfections of the c-fosCAT reporter construct with RasN17 (5 µg) into cardiomyocytes inhibited c-fos promoter activity induced by Et-1 (Fig. 6). However, H₂O₂ (25 µmol/l) treatment for 24 h on cardiomyocytes cotransfected with c-fosCAT and RasN17 (5 µg) led to 2.2-fold increase in CAT activity as compared to that of RasN17-transfected cells (Fig. 6B). All these results suggest that Ras mediate ROS generation and c-fos gene induction in cardiomyocytes exposed to Et-1.

View larger version (45K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Ras modulated intracellular ROS generation and c-fos mRNA induction. (A) Cardiomyocytes were transfected with either pSR-empty vector (5 µg), RasL61 (1 or 5 µg) or RasN17 (5 µg). Cells after transfection for 48 h were loaded with DCF-DA followed with Et-1 treatment and intracellular ROS were imaged by laser confocal microscopy. (B) Ras mediated c-fos mRNA levels induced by Et-1. Five micrograms of pSR-empty vector, RasN17, or RasL61 were transfected into cardiomyocytes for 48 h, followed by treating with 10 nmol/l Et-1 (lanes 2–4) or 25 µmol/l H2O2 (lanes 5 and 8) for 30 min. Some cells were treated with catalase (350 U/ml) (lanes 3 and 7) for 24 h. H2O2 treated cells were used as positive controls. c-fos mRNA levels were analyzed by using a densitometer and the results were normalized to those of 18S ribosomal RNA. Results are shown as mean±S.E.M. *P<0.05 vs. pSR-transfected control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. only RasN17-transfected cells. The experiment was repeated three times with reproducible results.

 

View larger version (57K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Ras mediated Et-1-induced c-fos promoter activities. (A) Fifteen micrograms of c-fosCAT reporter gene construct was cotransfected with either 5 µg of pSR-empty vector, RasN17, or RasL61 into cardiomyocytes for 24 h, followed by treating with 10 nmol/l Et-1 (lanes 2–4) or 25 µmol/l H2O2 (lanes 5 and 8). Some cells were pretreated with catalase (350 U/ml) (lane 3) before Et-1 treatment for 24 h. CAT2 (lane 9) and CAT3 (lane 10) are shown as positive and negative controls for CAT assay. Cells were harvested and CAT assays were performed as described in Section 2. (B) CAT activities were determined by normalizing percentage incorporation in each experimental group to that of β-galactosidase activities. Results are shown as mean±S.E.M. *P<0.05 vs. pSR-transfected control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. only RasN17-transfected cells; P<0.05 vs. only RasL61-transfected cells. The experiment was repeated three times with reproducible results.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In the present study, we demonstrated that neonatal rat cardiomyocytes exposed to Et-1 increased intracellular reactive oxygen species and resulted in an induction of c-fos gene expression via Ras pathway. Recent observation indicates that cells from various sources (i.e. endothelial cells, smooth muscle cells, etc.) exposed to various growth factors or stimuli generate ROS as second messengers [12]. Present study has clearly demonstrated that cardiomyocytes exposed to Et-1 increase intracellular ROS levels via Et A receptor-dependent mechanism. This finding is consistent with the previous report that the Et A is the main receptor type present in the rat heart [29]and cardiomyocytes [30]. Furthermore, the increase of ROS by Et-1 treatment was completely abolished by co-incubation of cardiomyocytes with the antioxidant enzyme catalase. This indicates that H₂O₂ is a major reactive oxygen species that contribute to the increase of c-fos induction in Et-1-treated cardiomyocytes. Catalase is a big molecule which is unlikely to diffuse into cells but appeared to rapidly clear the intracellularly Et-1-induced H₂O₂. Because H₂O₂ is relatively stable, the intracellular generated H₂O₂ that is permeable to membrane can be easily removed by catalase. Alternatively, the extracellular catalase may be uptake by the cardiomyocytes and results in a decrease of intracellular ROS levels. The uptake of catalase into cells and the decrease of intracellular ROS levels have been previously shown in platelet-derived growth factor (PDGF)-treated smooth muscle cells [31]. Catalase has also been effectively used in treating various cells to prevent from cytokine- or growth factor-induced gene expression [31–33]. This is also in agreement with our previous report demonstrating the inhibitory effect of catalase on hemodynamic forces-induced gene expression by reducing intracellular ROS levels [34–36]. The induction of ROS by Et-1 was also blocked by the glutathione (GSH) precursor NAC. NAC treatment raised the levels of intracellular GSH that might remove the intracellular H₂O₂ via GSH peroxidase [11]. Our ROS induction by Et-1 was a time- and dose-dependent phenomenon. The origin of this increased ROS is not clear. ROS, including H₂O₂, may be derived from the activated electron transfer reaction or other superoxide producing enzyme systems. How the cells respond to a stimulus and transduce early signals to ROS-producing machineries remain to be defined. Protein kinase C activation after Et-1 treatment may be involved in this process [37]. Nevertheless, our results clearly indicate that Et-1 treatment of cardiomyocytes induces ROS production via Et A receptor.

Changes in the redox status of cells are thought to be able to induce modification of cellular signaling molecules including various protein kinases, protein phosphatases and transcription factors [12]. In the present study, we used oxidant-inducible gene c-fos as a target gene to demonstrate that this Et-1-induced c-fos expression was an event involving intracellular redox changes. Et-1 treatment of cardiomyocytes, similar to H₂O₂ exposure, induced a transient increase of c-fos expression. This transient induction by Et-1 was inhibited by treatment of cardiomyocytes with catalase or NAC. As a positive control, cardiomyocytes treated with PMA stimulated their c-fos expression. PMA treatment of cardiomyocytes greatly increased intracellular ROS levels. However, PMA-induced c-fos expression was only partially inhibited by NAC or catalase pretreatment (data not shown). The immediate proto-oncogene c-fos is normally expressed at a minimal level in the mammalian heart. Increased proto-oncogene expression has been implicated in the development of cardiac hypertrophy [38]. Induction of c-fos, a transcription factor that interacts with cis-regulatory element AP-1 in many genes, has important consequences for downstream gene expression that may determine the phenotypic response to Et-1. The c-fos promoter region contains at least three cis-regulatory regions that are involved in the regulation of c-fos transcription; i.e., the SIE (sis-inducible element), AP-1 (Fos–Jun transcription factor complex) and the SRE (serum responsive element) [39]. The activation of c-fos transcription is thought to be mediated by the activation of MAPK [40]. It has also been demonstrated that H₂O₂ is capable of stimulating MAPK [41, 42]. In cardiomyocytes, Et-1 has been shown to activate two closely related MAPK isoforms, p42MAPK and p44MAPK [43]via a Ras-dependent pathway. Recent data even indicate that Ras is a common signaling target of ROS [44]. Thus, Et-1-induced ROS are likely to activate the signaling pathway that results in the induction of transcriptional activity of c-fos.

Our results further demonstrated that Ras was involved in Et-1-induced ROS generation because a proportional increase of ROS production was noticed as the transfection dose of RasL61 increased. Although we could not rule out the possibility that transfected cells might release some factor and then acting on other cells. However, the cardiomyocytes after RasL61-transfection did get brighter with fluorescence intensity. Accordingly, increased c-fos mRNA levels in RasL61-transfected cardiomyocytes were noticed. Furthermore, Et-1-induced c-fos mRNA level was attenuated by co-transfection of cardiomyocytes with RasN17. Consistently, c-fos promoter activities were increased by RasL61 and attenuated by RasN17 transfection. All these results strongly support that Ras is involved in the Et-1-induced ROS production that results in the increase of c-fos mRNA levels. These findings are consistent with those of previous studies indicating that Et-1 stimulates a Ras-based signaling cascade in mesangial cells [18]. Ras-dependent ROS production has also been shown in NIH 3T3 fibroblasts. Furthermore, expression of dominant negative Ras suppressed ROS production has been reported [20]. All these are consistent with our results and the premise that ROS are generated by Et-1 via a Ras dependent mechanism. Taken together, these results provide strong evidence for a Ras-based signaling cascade linking Et-1 receptors to ROS generation and the activation of the c-fos gene in cardiomyocytes.

We have previously demonstrated that ROS work as second messengers in vascular endothelial cells subjected to hemodynamic forces [34–36]. These increased ROS induced AP-1 binding to a cis-acting element (TRE) in the MCP-1 promoter region [34]and possible serum response element (SRE) site in the egr-1 promoter region [45]. Because c-fos promoter region contains AP-1 and SRE sites, it is possible that ROS modulate AP-1 and/or SRE binding via Ras-dependent signaling pathway. The detailed mechanisms as to how the ROS modulate the signaling pathway remain largely unknown and warrant further study. Nevertheless, this study demonstrates that ROS act as cellular signals in Et-1-treated cardiomyocytes. The important role of Et-1 for cardiac hypertrophy has been well documented [2, 3]. Because Et-1 induces ROS that result in c-fos induction which subsequently may regulate the expression of late genes, the suppression of intracellular redox changes by increasing antioxidant capability may provide some insight for preventing Et-1-induced gene alterations in cardiomyocytes during the development of cardiac hypertrophy. Improvement of myocardial redox status with vitamin E therapy that can attenuate the development of hypertrophy to heart failure has recently been reported [46]. Our results provide further support of the importance of ROS as second messenger for Et-1-induced responses in cardiovascular systems. This finding may offer clinical implications for the therapeutic potential of antioxidant treatment against the transition from compensatory hypertrophy to heart failure.

Time for primary review 28 days.

	Acknowledgements

 
The present work was partly supported by grant NSC 87-2314-B-002-229 from National Science Council, Taipei, Taiwan, R.O.C.

	Notes

 
¹ Present address: Faculty of Art and Science, University of Toronto, Toronto, Ontario, Canada.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Yanagisawa M., Kurihara H., Kimura S., et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (1988) 332:411–415.[CrossRef][Medline]
2. Wang D.L., Chen J.J., Shih N.L., et al. Endothelin stimulates cardiac - and β-myosin heavy chain gene expression. Biochem Biophys Res Commun (1992) 183:1260–1265.[CrossRef][Web of Science][Medline]
3. Yamazaki T., Komuro I., Kudoh S., et al. Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem (1996) 271:3221–3228.[Abstract/Free Full Text]
4. Shubeita H.E., McDonough P.M., Harris A.N., et al. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and gene expression in ventricular myocytes. J Biol Chem (1990) 265:13809–13817.[Abstract/Free Full Text]
5. Bogoyevitch M.A., Glennon P.E., Andersson M.B., et al. Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem (1994) 269:1110–1119.[Abstract/Free Full Text]
6. Bogoyevitch M.A., Ketterman A.J., Sugden P.H. Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes. J Biol Chem (1995) 270:29710–29717.[Abstract/Free Full Text]
7. Gille H., Sharrocks A., Shaw P. Phosphorylation of p62^TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature (1992) 358:414–417.[CrossRef][Medline]
8. Marais R., Wynne J., Treisman R. The SRF accessory protein ELS-1 contains a growth factor regulated transcription domain. Cell (1993) 73:381–393.[CrossRef][Web of Science][Medline]
9. Whitmarsh A.J., Shore P., Sharrock A.D., Davis R.J. Integration of MAP kinase signal transduction pathways at the serum response element. Science (1995) 269:403–407.[Abstract/Free Full Text]
10. Angel P., Imagawa M., Chiu R., et al. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell (1987) 49:729–739.[CrossRef][Web of Science][Medline]
11. Schreck R., Rieber P., Baeuerle P.A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kB transcription factor and HIV-1. EMBO J (1991) 10:2247–2258.[Web of Science][Medline]
12. Lander H.M. An essential role for free radicals and derived species in signal transduction. FASEB J (1997) 11:118–124.[Abstract]
13. Nagase T., Fukuchi Y., Jo C., et al. Endothelin-1 stimulates arachidonate 15-lipoxygenase activity and oxygen radical formation in the rat distal lung. Biochem Biophys Res Comm (1990) 168:485–489.[CrossRef][Web of Science][Medline]
14. Viani P., Zini I., Cervato G., Biagini G., Agnati L.F., Cestaro B. Effect of endothelin-1 induced ischemia on peroxidative damage and membrane properties in rat striatum synaptosomes. Neurochem Res (1995) 20:689–695.[CrossRef][Web of Science][Medline]
15. Kovacic-Milivojevic B., Zlock D.W., Gardner D.G. Ras inhibits jun-activated human atrial natriuretic peptide gene transcription in cultured ventricular myocytes. Circ Res (1997) 80:580–588.[Web of Science][Medline]
16. Takaya S., Nakafuku M., Kaziro Y. Function of Ras as a molecular switch in signal transduction. J Biol Chem (1992) 268:2244–2249.[Web of Science]
17. Thorburn A., Thorburn J., Chen S.Y., et al. The ras-dependent pathways can activate morphological and genetic markers of cardiac hypertrophy. J Biol Chem (1993) 268:2244–2249.[Abstract/Free Full Text]
18. Herman W.H., Simonson M.S. Nuclear signaling by endothelin-1. A Ras pathway for activation of the c-fos serum response element. J Biol Chem (1995) 270:11654–11661.[Abstract/Free Full Text]
19. Sundaresan M., Yu Z-X., Ferrans V.J., et al. Regulation of reactive oxygen species generation in fibroblasts by Rac1. Biochem J (1996) 318:379–382.[Web of Science][Medline]
20. Irani K., Xia Y., Zweier J.L., et al. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science (1997) 275:1649–1652.[Abstract/Free Full Text]
21. Neyses L., Nouskas J., Luyken J., et al. Induction of immediate-early genes by angiotensin II and endothelin-1 in adult rat cardiomyocytes. J Hypertension (1993) 11:927–934.[CrossRef][Web of Science][Medline]
22. Peng M., Huang L., Xie Z.J., Huang W.H., Askari A. Oxidant-induced activation of nuclear factor-kappa B and activator protein-1 in cardiac myocytes. Cellular Mol Biol Res (1995) 41:189–197.[Web of Science][Medline]
23. Chen J.J., Wang D.L., Shih N.L., Hsu K.H., Lien W.P., Liew C.C. Regulation of human cardiac myosin heavy chain genes: the effect of catecholamine. Biochem Biophys Res Commun (1992) 188:547–553.[CrossRef][Web of Science][Medline]
24. Deschamps J., Meijlink F., Verma I.M. Identification of a transcriptional enhancer element upstream from the proto-oncogene c-fos. Science (1985) 230:1174–1177.[Abstract/Free Full Text]
25. Feig L.A., Cooper G.M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol (1988) 8:3235–3243.[Abstract/Free Full Text]
26. Zhu H., Bannerberg G., Moldeus P., Shertzer H. Oxidation pathways for the intracellular probe 2',7'-dichlorofluorescin. Arch Toxicol (1994) 68:582–587.[CrossRef][Web of Science][Medline]
27. Curran T., Gordon M.B., Rubino K.L., Sambucetti L.C. Isolation and characterization of the c-fos(rat) cDNA and analysis of post-translational modification in vitro. Oncogene (1987) 2:79–84.[Web of Science][Medline]
28. Nose K., Shibanuma M., Kikuchi K., Kageyama H., Sakiyama S., Kuroki T. Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line. Eur J Biochem (1991) 201:99–106.[Web of Science][Medline]
29. Hilal-Dandan R., Merck D.T., Lujan J.P., Brunton L.L. Coupling of the type A endothelin receptor to multiple responses in adult rat cardiac myocytes. Mol Pharmacol (1994) 45:1183–1190.[Abstract]
30. Kanno K., Hirata Y., Tsujino M., et al. Up-regulation of ETB receptor subtype mRNA by angiotensin II in rat cardiomyocytes. Biochem Biophys Res Commun (1993) 194:1282–1287.[CrossRef][Web of Science][Medline]
31. Sundaresan M., Yu Z-X., Ferrans V.J., Irani K., Finkel T. Requirement for generation of H₂O₂ for platelet-derived growth factor signal transduction. Science (1995) 270:296–299.[Abstract/Free Full Text]
32. Ohba M., Shibanuma M., Kuroki K., Nose K. Production of hydrogen peroxide by transforming growth factor-1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol (1994) 126:1079–1088.[Abstract/Free Full Text]
33. Bae Y.S., Kang S.W., Seo M.S., et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide: Role in EGF receptor-mediated tyrosine phosphorylation. J Biol Chem (1997) 272:217–221.[Abstract/Free Full Text]
34. Wung B.S., Cheng J.J., Hsieh H.J., Shyy Y.J., Wang D.L. Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1. Circ Res (1997) 81:1–7.[Abstract/Free Full Text]
35. Hsieh H.J., Cheng C.C., Wu S.T., Chiu J.J., Wung B.S., Wang D.L. Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression. J Cell Physiol (1998) 175:156–162.[CrossRef][Web of Science][Medline]
36. Chiu J.J., Wung B.S., Hsieh H.J., Wang D.L. Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells. Arterio Throm Vas Biol (1997) 17:3570–3577.
37. Puceat M., Hilal-Dandan R., Strulovici B., Brunton L.L., Brown J.H. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. J Biol Chem (1994) 269:16938–16944.[Abstract/Free Full Text]
38. Barka T.H., van der Neon H., Shaw P.A. Proto-oncogene fos (c-fos) expression in the heart. Oncogene (1987) 1:439–443.[Web of Science][Medline]
39. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem (1995) 270:16483–16486.[Free Full Text]
40. Saklatvala J., Rawlinson L.M., Marshall C.J., Kracht M. Interleukin 1 and tumor necrosis factor activate the mitogen-activated protein (MAP) kinase kinase in cultured cells. FEBS Lett (1993) 334:189–192.[CrossRef][Web of Science][Medline]
41. Stevenson M.A., Pollock S.S., Coleman C.N., Calderwood S.K. X. X-Irradiation, phorbol esters and H₂O₂ stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates. Cancer Res (1994) 54:12–15.[Abstract/Free Full Text]
42. Guyton K.Z., Liu Y., Gorospe M., Xu Q., Holbrook N.J. Activation of mitogen-activated protein kinase by H₂O₂. J Biol Chem (1996) 271:4138–4142.[Abstract/Free Full Text]
43. Post G.R., Goldstein D., Thuerauf D.J., Glembotski C.C., Brown J.H. Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptor-induced hypertrophy in neonatal rat ventricular myocytes. J Biol Chem (1996) 271:8452–8457.[Abstract/Free Full Text]
44. Muller J.M., Cahill M.A., Rupec R.A., Baeuerle P.A., Nordheim A. Antioxidants as well as oxidants activate c-fos via Ras-dependent activation of extracellular-signal- regulated kinase 2 and Elk-1. Eur J Biochem (1997) 244:45–52.[Web of Science][Medline]
45. Datta R., Taneja N., Sukhatme V.P., Qureshi S.A., Weichselbaum R., Kufe D.W. Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation. Proc Natl Acad Sci USA (1993) 90:2419–2422.[Abstract/Free Full Text]
46. Dhalla A.K., Hill M.F., Singal P.K. Role of oxidative stress in transition of hypertrophy to heart failure. J Am Coll Cardiol (1996) 28:506–514.[Abstract]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				A. Laskowski, O. L. Woodman, A. H. Cao, G. R. Drummond, T. Marshall, D. M. Kaye, and R. H. Ritchie Antioxidant actions contribute to the antihypertrophic effects of atrial natriuretic peptide in neonatal rat cardiomyocytes Cardiovasc Res, October 1, 2006; 72(1): 112 - 123. [Abstract] [Full Text] [PDF]

				S. Javadov, D. Baetz, V. Rajapurohitam, A. Zeidan, L. A. Kirshenbaum, and M. Karmazyn Antihypertrophic Effect of Na+/H+ Exchanger Isoform 1 Inhibition Is Mediated by Reduced Mitogen-Activated Protein Kinase Activation Secondary to Improved Mitochondrial Integrity and Decreased Generation of Mitochondrial-Derived Reactive Oxygen Species J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1036 - 1043. [Abstract] [Full Text] [PDF]

				C.-H. Chen, T.-H. Cheng, H. Lin, N.-L. Shih, Y.-L. Chen, Y.-S. Chen, C.-F. Cheng, W.-S. Lian, T.-C. Meng, W.-T. Chiu, et al. Reactive Oxygen Species Generation Is Involved in Epidermal Growth Factor Receptor Transactivation through the Transient Oxidization of Src Homology 2-Containing Tyrosine Phosphatase in Endothelin-1 Signaling Pathway in Rat Cardiac Fibroblasts Mol. Pharmacol., April 1, 2006; 69(4): 1347 - 1355. [Abstract] [Full Text] [PDF]

				Y. J. Kang Cardiac Hypertrophy: A Risk Factor for QT-Prolongation and Cardiac Sudden Death Toxicol Pathol, January 1, 2006; 34(1): 58 - 66. [Abstract] [Full Text] [PDF]

				T.-H. Cheng, N.-L. Shih, S.-Y. Chen, J.-W. Lin, Y.-L. Chen, C.-H. Chen, H. Lin, C.-F. Cheng, W.-T. Chiu, D. L. Wang, et al. Nitric Oxide Inhibits Endothelin-1-Induced Cardiomyocyte Hypertrophy through cGMP-mediated Suppression of Extracellular-Signal Regulated Kinase Phosphorylation Mol. Pharmacol., October 1, 2005; 68(4): 1183 - 1192. [Abstract] [Full Text] [PDF]

				H-H Chao, J-J Chen, C-H Chen, H Lin, C-F Cheng, W-S Lian, Y-L Chen, S-H Juan, J-C Liu, J-Y Liou, et al. Inhibition of angiotensin II induced endothelin-1 gene expression by 17-{beta}-oestradiol in rat cardiac fibroblasts Heart, May 1, 2005; 91(5): 664 - 669. [Abstract] [Full Text] [PDF]

				S.-H. Juan, J.-J. Chen, C.-H. Chen, H. Lin, C.-F. Cheng, J.-C. Liu, M.-H. Hsieh, Y.-L. Chen, H.-H. Chao, T.-H. Chen, et al. 17{beta}-Estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1254 - H1261. [Abstract] [Full Text] [PDF]

				H.-J. Hong, P. Chan, J.-C. Liu, S.-H. Juan, M.-T. Huang, J.-G. Lin, and T.-H. Cheng Angiotensin II induces endothelin-1 gene expression via extracellular signal-regulated kinase pathway in rat aortic smooth muscle cells Cardiovasc Res, January 1, 2004; 61(1): 159 - 168. [Abstract] [Full Text] [PDF]

				Y. Garnier, A. B. C. Coumans, A. Jensen, T. H. M. Hasaart, and R. Berger Infection-Related Perinatal Brain Injury: The Pathogenic Role of Impaired Fetal Cardiovascular Control Reproductive Sciences, December 1, 2003; 10(8): 450 - 459. [Abstract] [PDF]

				J.-C. Liu, J.-J. Chen, P. Chan, C.-F. Cheng, and T.-H. Cheng Inhibition of Cyclic Strain-Induced Endothelin-1 Gene Expression by Resveratrol Hypertension, December 1, 2003; 42(6): 1198 - 1205. [Abstract] [Full Text] [PDF]

				T.-H. Cheng, P.-Y. Cheng, N.-L. Shih, I.-B. Chen, D. L. Wang, and J.-J. Chen Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts J. Am. Coll. Cardiol., November 19, 2003; 42(10): 1845 - 1854. [Abstract] [Full Text] [PDF]

				C.-M. Cheng, H.-J. Hong, J.-C. Liu, N.-L. Shih, S.-H. Juan, S.-H. Loh, P. Chan, J.-J. Chen, and T.-H. Cheng Crucial Role of Extracellular Signal-Regulated Kinase Pathway in Reactive Oxygen Species-Mediated Endothelin-1 Gene Expression Induced by Endothelin-1 in Rat Cardiac Fibroblasts Mol. Pharmacol., May 1, 2003; 63(5): 1002 - 1011. [Abstract] [Full Text] [PDF]

				D.-w. Jeong, M.-H. Yoo, T. S. Kim, J.-H. Kim, and I. Y. Kim Protection of Mice from Allergen-induced Asthma by Selenite. PREVENTION OF EOSINOPHIL INFILTRATION BY INHIBITION OF NF-kappa B ACTIVATION J. Biol. Chem., May 10, 2002; 277(20): 17871 - 17876. [Abstract] [Full Text] [PDF]

				T. Suzuki and T. Miyauchi A novel pharmacological action of ET-1 to prevent the cytotoxicity of doxorubicin in cardiomyocytes Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2001; 280(5): R1399 - R1406. [Abstract] [Full Text] [PDF]

				V. J. Thannickal and B. L. Fanburg Reactive oxygen species in cell signaling Am J Physiol Lung Cell Mol Physiol, December 1, 2000; 279(6): L1005 - L1028. [Abstract] [Full Text] [PDF]

				J. Fei, C. Viedt, U. Soto, C. Elsing, L. Jahn, and J. Kreuzer Endothelin-1 and Smooth Muscle Cells : Induction of Jun Amino-Terminal Kinase Through an Oxygen Radical-Sensitive Mechanism Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1244 - 1249. [Abstract] [Full Text] [PDF]

				D. R. Pimentel, J. K. Amin, L. Xiao, T. Miller, J. Viereck, J. Oliver-Krasinski, R. Baliga, J. Wang, D. A. Siwik, K. Singh, et al. Reactive Oxygen Species Mediate Amplitude-Dependent Hypertrophic and Apoptotic Responses to Mechanical Stretch in Cardiac Myocytes Circ. Res., August 31, 2001; 89(5): 453 - 460. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Cheng, T.H. Articles by Chen, J.J. Search for Related Content PubMed PubMed Citation Articles by Cheng, T.H. Articles by Chen, J.J. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics